Cancer stem cells, the key population driving growth and metastasis of triple-negative breast cancer and other breast carcinomas, exist in states with epithelial and mesenchymal phenotypes. Mesenchymal breast cancer stem cells drive local invasion and metastatic spread, while cancer stem cells transition to an epithelial state to proliferate in primary and metastatic sites. Reversible transitions from epithelial to mesenchymal (EMT) and mesenchymal to epithelial (MET) states of cancer stem cells drive tumor progression, underscoring the critical need to understand mechanisms driving these transitions in tumor environments. We hypothesize that extracellular matrix proteins and mechanical stress regulate epithelial and mesenchymal transitions of triple negative breast cancer stem cells in distinct compartments in the metastatic cascade: primary tumor, intravascular, and bone/bone marrow metastases. To systematically interrogate this hypothesis, we will employ a physical sciences approach based on precisely tunable tissue-engineered tumor environments pioneered by our group. We have developed a novel polymeric scaffold architecture that maintains functional conformations of key extracellular matrix proteins present in primary breast tumors while independently controlling elastic modulus of the engineered primary tumor environment. Preliminary studies show these scaffolds control EMT and MET phenotypes of human breast cancer cell lines and cancer cells obtained directly from patients. To analyze EMT and MET states of cancer cells in the vasculature, we will use an innovative, label-free microfluidic device to capture circulating tumor cells. We also will subject circulating tumor cells to a recently identified intravascular mechanical and chemical stress, neutrophil extracellular traps (NETs), using a new technology we developed to artificially reproduce NETs in vascular mimetic microchannels. Finally, we will simulate components of the bone/bone marrow metastatic environment in vivo using implanted scaffolds with osteogenic cells and mesenchymal stromal cells. For all aspects of this research, we will image dynamics of EMT and MET plasticity with a newly designed fluorescent reporter system. We will quantify effects of engineered tumor environments on EMT and MET states of breast cancer stem cells through these specific aims: 1) investigate effects of extracellular matrix proteins and mechanical stress on stem cell plasticity in a primary tumor; 2) establish dynamics of EMT and MET in circulating tumor cells interacting with NETs; and 3) determine cell-environmental interactions driving transitions of cancer stem cells metastatic to bone. Through an integrated, multi-disciplinary approach, we will establish functions of physical components of tumor environments on plasticity of triple-negative breast cancer stem cells, which we expect will lead to new treatment options to target this aggressive disease.